Direct induction of T lymphocyte-specific gene expression by the mammalian Notch signaling pathway

Abstract

The Notch signaling pathway regulates the commitment and early development of T lymphocytes. We studied Notch-mediated induction of the pre-T cell receptor alpha (pTa) gene, a T-cell-specific transcriptional target of Notch. The pTa enhancer was activated by Notch signaling and contained binding sites for its nuclear effector, CSL. Mutation of the CSL-binding sites abolished enhancer induction by Notch and delayed the up-regulation of pTa transgene expression during T cell lineage commitment. These results show a direct mechanism of stage- and tissue-specific gene induction by the mammalian Notch/CSL signaling pathway.

Figure 1

CSL-binding sites in the pTa enhancer. (A) Alignment of the mouse (m) and human (h) enhancer core sequences. Previously identified binding sites are boxed, and the potential CSL-binding sites are highlighted as shaded boxes. The mutations introduced into CSL sites are shown next to the sites. (B) The binding of CSL to sites from the pTa enhancer. EMSA was performed with radio-labeled oligonucleotide probes, and the resulting CSL–DNA complexes are indicated by an arrow. The probes included CSL-binding sites from the Hes1 promoter (HES), the mouse pTa enhancer (mE), and the 5′ and 3′ sites from the human pTa enhancer (hE5′ and hE3′, respectively). The corresponding probes harboring the mutations shown in A are indicated by an asterisk (mE*, hE5′*, and hE3′*). (Left panel) In vitro translated CSL or a control translation reaction (–) were incubated with the mE probe in the presence of anti-FLAG Ab or a control Ab (ctrl.). (Middle panel) In vitro translated CSL was incubated with the indicated probes. (Right panel) Nuclear extract from BW5147 cells was incubated with the mE probe in the presence of 10-fold and 100-fold molar excess of the indicated unlabeled competitor probes (C).

Figure 2

Induction of the pTa enhancer by NICD. Cell line 293 was transfected with reporter constructs together with the NICD expression vector. Normalized reporter activities in the presence of NICD or of an empty expression vector were determined, and their ratio is presented as fold induction by NICD. The results represent the mean + SD of three independent experiments. Reporter constructs contained the indicated enhancer fragments upstream of the mouse pTa promoter (pTa) or of the SV40 promoter TATA box (TATA). The mouse (mpTa) or human (hpTa) pTa enhancers were either wild-type or harbored the mutations shown in Figure 1A (mpTa mut., hpTa mut.).

Figure 3

Notch-mediated induction and intrinsic activity of the pTa enhancer fragments. Cells were transfected with reporter constructs containing the indicated human pTa enhancer fragments upstream of the TATA box. The CSL-binding sites (positions 188–95 and 224–31) are shown as shaded bars, and asterisks indicate mutations in these sites as shown in Figure 1A. The results represent the mean + SD of three independent experiments. (A) The induction of the pTa enhancer fragments by NICD in 293 cells was determined as in Figure 2. (B) The relative activity of the pTa enhancer fragments in an immature T cell line, LR1, was determined as the ratio of reporter activity to the activity of an enhancerless reporter vector.

Figure 4

Generation of transgenic reporter mice to examine the function of CSL sites in vivo. (A) Strategy for modification of the human pTa locus by homologous recombination in bacteria. The coding regions, UTR, and the upstream enhancer of pTa are shown as black, white, and vertical gradient boxes, respectively. Prokaryotic selection markers (Cm^r and Zeo^r) are indicated by gray boxes. The EGFP gene (elliptical gradient box) and polyA signal (pA, white box) were introduced into the first pTa exon within the genomic BAC clone. Subsequently, mutations of both CSL sites (vertical bars) were introduced into the pTa enhancer. After the recombination and Flp-mediated excision, an Alu repeat 3′ to the enhancer was replaced by a single FRT site (black triangle). The resulting constructs contained either the wild-type (EGFP–Ewt) or a mutated (EGFP–Emut) enhancer sequence. (B) Expression of the modified pTa locus in transgenic mice. Shown are histograms of EGFP fluorescence in the indicated lymphocyte subsets of an EGFP–Ewt transgenic mouse (shaded traces) and a control nontransgenic mouse (empty traces). (Left panel) Thymocyte subsets were defined as follows: DN (CD3⁻CD4⁻CD8⁻), ISP (CD3⁻CD4⁻CD8⁺), large DP (large CD3⁻CD4⁺CD8⁺), small DP (small CD3^−/lowCD4⁺CD8⁺) and SP (CD3^highCD4⁺CD8⁻ and CD3^highCD4⁻CD8⁺). (Right panel) Other lymphocyte subsets included splenic T cells (CD3^high), NK cells (NK1.1⁺), splenic B cells (B220⁺CD3⁻), bone marrow (BM) B cells (lin⁺B220⁺), and bone marrow lineage marker-negative cells (lin⁻B220⁻).

Figure 5

EGFP reporter expression in the earliest thymocyte subpopulations. (A) Thymocytes were stained with a cocktail of mAb to lineage markers, and the lineage-negative thymocytes (1%–2% of the total) were gated on the basis of CD44 and CD25 expression as shown on the density plot at the left. Shown are the histograms of the EGFP fluorescence in the gated DN thymocyte subsets (DN1–DN4) from a control nontransgenic mouse or from representative transgenic mice. The logarithmic scale was used on the Y axis to facilitate comparison between the different subsets. (B) Mice from three independent transgenic lines of each type were stained as above, and the percentage of EGFP-positive cells was determined in each DN subset. Control wild-type thymocytes contained <0.5% positive cells in each subset. The representative staining results are shown as pairwise comparisons between the EGFP–Ewt (open circles) and EGFP–Emut (filled circles) lines with similar levels of overall EGFP expression. (C) Adult or 16.5 days postcoitum (dpc) fetal thymocytes from the same transgenic lines as in A were stained with mAb to the lineage markers, CD25 and either CD44 or CD117. Shown are contour plots of EGFP versus CD117 or CD44 fluorescence in lineage-negative CD25⁺ cells encompassing subsets DN2 and DN3. Horizontal lines show the baseline EGFP fluorescence in nontransgenic control mice, and vertical dashed lines define the CD44/CD117^high (DN2) subset.